Tea is a popular caffeine containing beverage with anti-cancerous properties (Jankun, J., Selman, S. H., Swiercz, R. Why drinking green tea could prevent cancer. Nature 5:561; 1997). Tea is also an important employment generator and a major foreign exchange earner in all the tea growing areas of the world (Wilson, K. C. Botany and Plant Improvement In: Wilson R. C., ed. Coffea, Cocoa and Tea. CABI Publishing, Wallingford, UK: 167–173; 1999). While, the total production of tea is not sufficient enough to meet the demands of the domestic and the world markets (Kabra, G. D. Tea statistics for 1999 In: Tea time, Vol. VIII, No. 3 September–November 1999, 30–31; 1999). The yield and quality of tea is further reduced by different biotic (fungi, pests and viruses) and abiotic (frost, hail, chilling, drought, nutritional deficiencies etc.) stresses (Wilson, K. C. Botany and Plant Improvement In: Wilson R. C., ed. Coffea, Cocoa and Tea. CABI Publishing, Wallingford, UK: 167–173; 1999). Although for most crops, higher yield per unit area is of primary importance but the major objective for tea is improved yield coupled with better adaptibility and cup characters. Moreover, the world market has critical standards for tea from different parts of the world to which the products must conform in order to attain high commercial value.
Superior stress resistant tea plants combining both yield and cup quality are therefore, of utmost importance (Barua, D. N. The tea plant of commerce In: Barua, D. N., ed. Science and practice in tea culture, Tea Research Association Calcutta; 53–68; 1989). Crop improvement programmes also aim at reasonable degree of morphological homogeneity in the progeny. Long life cycles of almost 10 years coupled with high degree of self incompatibility and inbreeding depression (Barua, D. N. The tea plant of commerce In: Barua, D. N., ed. Science and practice in tea culture, Tea Research Association Calcutta; 53–68; 1989) are the major limitations for conventional tea breeding programmes. The important and efficient alternative for overcoming these limitations is genetic transformation through Agrobacterium tumefaciens or biolistic wherein desired genes can be directly introduced into the plant genome.
Biolistic has been successfully employed in the genetic improvement of woody perennials specially when the plants have long life cycle or when the basic information about plant inheritance is lacking. Thus genetic transformation through biolistic holds a tremendous potential in tea specially when the leaves are used as the initial explants. More so, because leaf explants despite having a tremendous potential for crop improvement are highly recalcitrant to Agrobacterium tumefaciens-mediated transformation probably due to the presence of certain phenolics (Biao Xi, Toru K, Jian Xu, Yongyan B Effect of polyphenol compounds in tea transformations. Abstr. no. 314. In: American Society of Plant Physiologists, Plant Biology 1998).
Although some tea clones have been identified which are high yielding as well as of high quality, yet these are susceptible to blister blight disease. Biotechnological improvement through homogenous tissues like leaf explants is required in these clones because heterogeneous tissues like cotyledon explants would result in genetic variability and loss of the desirable character of high yield and good quality. Therefore, use of leaf explants was important. However, the transformation of leaves through Agrobacterium tumefaciens is known to be in effective due to high content of certain polyphenols.
It was realized that primarily three factors viz. (i) increased surface area for maximum particle penetration, (ii) minimum cell damage/injury and (iii) maximum regeneration efficiency were required in order to make the transgenic protocol successful. Therefore, a method for biolistic mediated transgenic production of tea (Camellia sinensis (L.) O. Kuntze) using leaf explants was developed taking into account the above three factors in order to enable further genetic improvement of selected elites.
Genetic transformation through Agrobacterium tumefaciens was first initiated in tea leaves (Matsumoto S and Fukai M 1998 Agrobacterium tumefaciens mediated gene transfer in tea plant (Camellia sinensis) cells. Japan Agricultural Research Quarterly, 32: 287–291; Matsumoto S and Fukai M 1999 Effect of acetosyringone application on Agrobacterium mediated gene transfer in tea plant (Camellia sinensis), Bulletin of the National Research Institute of vegetables, ornamental plants and tea, Shizuoka, Japan, 14: 9–15) wherein transformed leaf callus was produced using 500 μM Acetosyringone was selected at 200 μg/ml kanamycin. These transformed calli showed PCR amplification for nptII gene primers. The major draw back is that transgenic plants could not be regenerated from these transformed leaf calli. Even induction of callus on the leaves required a very high dose of the costly chemical Acetosyringone.
Genetic transformation through Agrobacterium tumefaciens has also been attempted by Biao (Biao Xi, Toru K, Jian Xu, Yongyan B Effect of polyphenol compounds in tea transformations. Abstr. no. 314. In: American Society of Plant Physiologists, Plant Biology 1998) wherein leaf and cotyledons were tested. The draw back of the report is that the leaf explants could not be significantly infected with Agrobacterium tumefaciens and could not be transformed because of high content of phenolics mainly catechins.